Specific steps and molecular mechanisms involved in calcium uptake will be compared in sarcoplasmic reticulum isolated from normal and failed hearts in order to identify the cause of the depression of calcium uptake activity observed in cardiac failure. Failure will be induced by ischemia, endotoxin or pressure overload. These three types of failure will be compared to determine to what extent a common mechanism is involved in producing failure. Highly purified sarcoplasmic reticulum isolated by density augmentation or isopycnic centrifugation will be used. The partial reactions of the ATPase and their coupling to calcium transport will be studied in relation to calcium uptake capacity. Parallel studies will be carried out to determine the molecular basis for the change in calcium accumulating capacity. The role of phospholipids will be analyzed by phospholipid substitution. The molecular organization of the (Ca plus Mg) ATPase will be studied by analyzing cooperativity in ATP binding and by analyzing the topology of ATP binding to active and effector sites. 8-Azido-ATP will be used to label the active and effector sites.